Progresses and challenges in Strongyloides spp. proteomics

The availability of high-quality data of helminth genomes provided over the past two decades has supported and accelerated large-scale ‘omics studies and, consequently, the achievement of a more in-depth molecular characterization of a number of pathogens. This has also involved Strongyloides spp. and since their genome was made available transcriptomics has been rather frequently applied to investigate gene expression regulation across their life cycle. Strongyloides proteomics characterization has instead been somehow neglected, with only a few reports performing high-throughput or targeted analyses associated with protein identification by tandem mass spectrometry. Such investigations are however necessary in order to discern important aspects associated with human strongyloidiasis, including understanding parasite biology and the mechanisms of host–parasite interaction, but also to identify novel diagnostic and therapeutic targets. In this review article, we will give an overview of the published proteomics studies investigating strongyloidiasis at different levels, spanning from the characterization of the somatic proteome and excretory/secretory products of different parasite stages to the investigation of potentially immunogenic proteins. Moreover, in the effort to try to start filling the current gap in host-proteomics, we will also present the first serum proteomics analysis in patients suffering from human strongyloidiasis. This article is part of the Theo Murphy meeting issue ‘Strongyloides: omics to worm-free populations’.


Human strongyloidiasis and other Strongyloides spp.
Human strongyloidiasis by Strongyloides stercoralis is widely distributed across the world, with foci of current intense transmission in areas of poor sanitation and inadequate wastewater disposal [1].Less prevalent species causing human infection are known to be endemic in Papua New Guinea (Strongyloides fuelleborni kellyi) and in parts of Africa (Strongyloides fuelleborni fuelleborni) [2].
Non-human primates, dogs and wild canids (and probably cats) can also be infected by S. stercoralis [3], thus the zoonotic potential is under investigation.Other animals may have distinct Strongyloides species; among the latter S. ratti and S. venezuelensis, parasites of rodents, have been widely used in laboratory experimental infections [4] both for research purposes and to retrieve crude extracts as antigens for the implementation of serological assays for the diagnosis of human strongyloidiasis [5].
Acute infection is seldom observed in tourists returning from endemic areas [6].Clinical manifestations correlate with the larval migration across the body: immediately after skin penetration, infected individuals may present cutaneous signs and symptoms such as rash, pruritus and oedema at the portal of entry of the filariform infective larvae.Loeffler syndrome, characterized by cough and dyspnoea, follows a few days later, sometimes along with urticarial rash; eosinophilia is typically observed.Two-three weeks from infection, following the settlement of adult female worms in the gut, acute gastrointestinal symptoms (e.g.diarrhoea, abdominal pain) may occur [6,7].
Chronic infection, due to the autoinfective cycle, can lead to a wide range of clinical manifestations, from unapparent disease to severe systemic syndromes.Similarly to acute infection, symptoms usually involve skin, respiratory tract and intestine [2].Eosinophilia is frequently observed, although the eosinophil count can be within the normal range or fluctuating [2].Hostparasite interaction is usually regulated in immunocompetent individuals, while immunosuppression leads to an uncontrolled, accelerated autoinfection cycle, resulting in a dramatic increase in parasite load.This can cause mechanical obstruction with consequent worsening of clinical conditions (e.g.paralytic ileus, respiratory impairment); moreover, sepsis and/or meningitis by Gram negative bacteria have been associated with hyperinfection, contributing to increase the fatality rate.Further, larvae and even other parasite stages (adults and eggs) can disseminate all over the body.Risk factors that have been frequently associated with the development of hyperinfection/ dissemination are human T-cell lymphotropic virus type1 (HTLV-1) infection, malignancies, treatment with steroids and other immunosuppressant drugs (such as biologics) [8].
Strongyloides stercoralis has a complex life cycle characterized by the alternation between free-living and parasitic life stages, in addition to its peculiar ability to undergo autoinfection within the human host [9].The ability of this parasite to generate chronic long-lasting infections usually without apparent clinical manifestations [10] clearly indicates the establishment and efficient regulation of the interaction between the host and the parasite that should be discerned in order to achieve a more comprehensive knowledge of the pathophysiological mechanisms associated with human strongyloidiasis.Strongyloides, similar to most other helminths, is known to trigger a Th2 immune response, which should protect against hyperinfection.This response is associated with the production of type 2 cytokines and reduced Th1 and Th17 responses [2].However, this classical type 2 profile was not observed in a cohort of elderly Italians with chronic strongyloidiasis lasting for decades and in the absence of re-exposure [11].Mechanisms of disease tolerance might in fact establish during chronic strongyloidiasis as a defence tool for the host to limit damage associated with a persistently activated immune response.At the same time, the parasite could exploit this tolerance to ensure its survival within the host [12][13][14].Although the specific mechanisms are still largely unknown, it is thus clear that the host-parasite interplay is crucial in the maintenance of parasitism during strongyloidiasis.

Insights into the somatic proteome of
Strongyloides spp.
Compared to other helminth parasites, proteomics investigations of Strongyloides spp.have been largely under-reported.Recent technological advances in the field of proteomics offer the possibility of employing different strategies to achieve an adequate protein identification even from complex samples, including from multi-cellular organisms like helminths.These strategies encompass (i) the classical in-gel separation methods coupled with band/spot excision, in-gel digestion and subsequent protein identification; (ii) the analysis of protein suspensions by tandem mass spectrometry (LC-MS/MS) possibly, but not necessarily, coupled with different fractionation (e.g.off-gel electrophoresis, strong cation exchange) and/or labelling methods.The more recent advent of data-independent acquisition (DIA) mass spectrometry [15] is currently allowing the quantitative analysis of low-abundant samples without the need for in-gel protein separation, even though such an approach has yet to be applied to the study of Strongyloides spp.The first report describing the proteomics analysis of S. stercoralis infective larvae (iL3) somatic proteome was published in 2010, when Marcilla and colleagues employed a controlled proteolytic cleavage to identify proteins associated with the parasite surface as well as from whole larval lysate [16].Although the number of identifications (i.e. 26 proteins) was limited by the lack of a reference genome, the different digestion treatments performed in the study allowed the identification of proteins potentially associated with the larval surface, since they were detected after short digestion of whole larvae with trypsin.Importantly, some of the identified proteins-such as galectins, 14-3-3 and heat shock proteins-were subsequently found to be associated with S. venezuelensis iL3 somatic proteome and with S. ratti excretory/secretory products (ESPs) (discussed in the next paragraph) [17,18].A more comprehensive coverage of the somatic proteome was indeed later obtained for S. ratti parasitic and free-living adult females [19] and for S. venezuelensis iL3 [18], taking advantage of the availability of a reference genome for S. ratti and of a draft genome for S. venezuelensis [19].The tandem mass spectrometry approach applied to the study of S. ratti provided a catalogue encompassing 1266 proteins from adult females.A comparative analysis revealed extensive differences in the protein expression between free living (Ff ) and parasitic female (Pf ) worms, since 569 proteins were reported as upregulated in Pf and 409 in Ff.Even though the number of differential proteins was particularly elevated (maybe due to the applied statistics) and similar differences were not observed at the transcriptome level [19], the study clearly showed the complexity of an in depth proteomics characterization of helminth parasites.Indeed, in order to comprehensively characterize these parasites, the different life stages and the different sub-proteomes should be investigated and ideally compared.
A large number of somatic proteins (i.e.877) was also identified from the shot-gun proteomics analysis of S. venezuelensis iL3.The most abundant components of S. venezuelensis iL3 somatic proteome included enzymes (particularly oxidoreductases), structural proteins, CAP domain proteins, heat shock proteins, proteases and protease inhibitors [18].It is noteworthy that some of these proteins were proposed to be potentially involved in Strongyloides parasitism in comparative genomics and transcriptomics analyses [19,20].Very similar results were recently obtained by our group through the analysis of S. stercoralis iL3 proteome [21].Indeed, we identified 430 proteins from S. stercoralis iL3 isolated from a clinical sample.Through the use of a semi-automated strategy, we annotated our dataset for gene ontology terms and protein domains, including uncharacterized proteins that represented 43% of our identifications.In agreement with previous reports, we confirmed the high representation within iL3 somatic proteome of proteins potentially associated with Strongyloides parasitism, including oxidoreductases, peptidases and peptidase inhibitors, proteins containing SCP/TAPS/CAP or thioredoxin domains and proteins belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families, galectins and transthyretin-like proteins [21].Importantly, we royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20220447 also identified with good confidence Ss-NIE, a protein employed in several in-house and commercial kits for the serodiagnosis of human strongyloidiasis [22][23][24][25].
Taken together, these proteomic catalogues could contribute to building a core proteome shared between Strongyloides species and/or life stages.Such analyses should thus be expanded in order to cover different life stages (especially the parasitic ones) and the different species (table 1).

Proteomics analysis of Strongyloides spp. excretory/secretory products
Helminth parasites have developed different mechanisms in order to survive and to adapt to the adverse conditions within their hosts.It is now evident that the release of ESPs plays a central role in this host-parasite interaction [33].ESPs include both waste molecules excreted in the environment and biomolecules actively secreted through specific pathways by live helminths [34].Since they are active at the host-parasite interface and in the parasite-mediated modulation of the host immune system [33,35], ESPs represent a unique source of potential targets for the development of diagnostic tools and vaccines, as well as for therapeutic interventions for other pathological conditions, including auto-immune disorders [36][37][38].For these reasons, they are raising significant interest within the scientific community and the release of ESPs has already been proven and investigated in multiple helminth species, including hookworms [39], Anisakis spp.[40], whipworms [41], filarial nematodes [42] and Strongyloides spp.
ESPs include a variety of biomolecules, i.e. small molecules, lipids and carbohydrates [39]; however, a predominant functional role is played by proteins.The prediction of secreted proteins using bioinformatic tools is often incomplete, as it is now clear that mechanisms of unconventional protein secretion exist and only a limited portion of secreted proteins possess the signal peptides driving the classical endoplasmic reticulum-Golgi secretion pathway [43].The experimental investigation of ESP protein composition using high-throughput approaches is thus essential to gain novel insights into the immunomodulatory properties of parasite secretome.
As mentioned above, ESPs are also being investigated in Strongyloides spp. to reveal novel aspects of parasite biology as well as their immunomodulatory properties.Nonetheless, apart from a few studies dating back to the '80s and '90s [44,45], ESPs from S. stercoralis are yet to be investigated in depth using up-to-date high throughput approaches.More pieces of evidence have instead been collected using the close relatives S. venezuelensis and S. ratti (table 1).
A comparative proteomics analysis revealed a stagedependent protein composition of S. ratti ESPs since different in-gel protein profiles were observed, further confirmed by protein identification by tandem mass spectrometry.In particular, L3 infective larvae (iL3) showed the highest number of stage-specific proteins (n = 196) compared to Pf (n = 79) and free-living larvae (Fl, n = 35) [17].It is worth mentioning that those iL3 had not been exposed to a host, thus they were presumably developmentally arrested.Given that protein release within ESPs was proven to occur via an excretion/ secretion process and not as a consequence of cell death or damage [17], the presence of proteins specific to iL3 ESPsincluding proteases-might support their role in the establishment of the infection or in the progression throughout the parasite life cycle.Nonetheless, it would be particularly interesting to explore and compare ESP proteins released by iL3 external to the host or at the very early stage of host invasion.Such analysis could in fact contribute in revealing additional factors specifically associated with iL3 mechanisms of infection and parasitism.Beside stage-specific proteins, 23.8% of the identified proteins were common to the three life stages analysed, including heat-shock proteins, galectins, enzymes, fatty acid binding protein and structural proteins [17].These shared proteins could contribute to building a core ESP proteome from Strongyloides spp., even though the variability in larval maintenance conditions across studies should be considered as a possible bias of these analyses.
The untargeted proteomics characterization was also performed on ESPs released by S. venezuelensis iL3 and Pf [28].Some iL3 ESP proteins had already been proposed to be potentially associated with parasitism in S. stercoralis genomic and transcriptomics analyses [19], including astacins, astacin-like peptidases and SCP/TAPS proteins.Indeed, although their molecular function is still largely unknown, these proteins have been proposed to be involved in parasite migration through the host as well as in immunomodulation [46,47].
Younis et al. used tandem mass spectrometry to investigate specific protein categories, namely small heat shock proteins (sHSPs), within ESPs of S. ratti parasitic female worms.Through an extensive bioinformatics analysis, they assigned two sequences as putative HSP-17 (then named Sra-HSP-17.1 and Sra-HSP-17.2), which were further characterized to establish their biological role in the host-pathogen interaction [26].Indeed, Sra-HSP-17s were found to be highly immunogenic in immunized rats as well as being recognized by the antibodies present in S. stercoralis-infected patients [26].
An important component of helminth-derived ESPs is represented by extracellular vesicles (EVs or exosome-like vesicles).Evidence supporting the active release of EVs from different helminth species both in vitro and in vivo has now been collected [48], however EV release in Strongyloides has so far been neglected.These vesicular elements carrying biological material are key players in inter-parasite and hostpathogen interactions [48][49][50][51].Described for the first time in 2012 in the trematodes Echinostoma caproni and Fasciola hepatica [52], they have subsequently raised large interest within the helminth scientific community and helminth-derived EVs have now been described in at least 17 helminth species [53].In parallel, the characterization of their protein cargo has become of importance in order to reveal novel potential biomarkers for disease diagnosis and control [53,54].
Despite their potential, the study of helminth-derived EVs presents numerous challenges and a standardization of the working procedures was recently deemed necessary, prompting the publication of recommended guidelines for their study [55].Although experimental data documenting Strongyloides-derived EVs are not yet available, Gonzales et al. employed bioinformatics tools to predict the presence of numerous proteins secreted within EVs among those identified in S. venezuelensis iL3 ESPs [27], concluding that EVs release is also likely to occur in Strongyloides.

Immunoproteomics
The ultimate goal of most proteomics studies applied to Strongyloides spp., performed either on the somatic proteome royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20220447 The number of proteins identified with at least two unique peptides is 766.
b Overall number of proteins identified using different experimental conditions. c Protein identification analysis only focussed on HSP proteins.
d For this category, the number of identified proteins corresponds to those identified from immuno-reactive bands/spots or predicted in silico.
royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20220447 or on the secretome, is the identification of protein targets for the development of novel serological assays.Indeed, although serological assays represent the most accurate diagnostic methods among those available, their performance still needs to be improved, both as screening and as diagnostic tools [56].Compared to classical parasitological methods, they present lower specificity also due to possible cross-reactions with other helminths [5].The sensitivity of serological methods can instead be very high (up to 83-95%), even though it can decrease in cases of immunosuppression or at the very early stage of infection [5].Another important issue associated with strongyloidiasis serodiagnosis is the possibility of false positive results due to a delay in seroreversion after treatment.The identification of markers whose detection is specifically indicative of an active infection would be particularly useful in developing novel diagnostic tests allowing this issue to be overcome.It is thus clear that current diagnostic methods need to be improved to achieve more reliable disease management and control.Two main paths have been undertaken by the scientific community to try to improve current serological tests using immunoproteomics approaches: (1) the identification of antigenic fractions from S. stercoralis, S. venezuelensis or S. ratti for the development of serological assays based on homologous or heterologous crude extracts; (2) the identification of specific S. stercoralis immunogenic proteins to be used in recombinant antigen-based tests (table 1).Both approaches have their own advantages and disadvantages.Indeed, although they are easily obtainable, since crude extracts rely on the use of host laboratory animals for their supply, they are associated with inter-batch variability and usually present a higher level of cross-reaction with other helminthiases compared with recombinant proteins.On the other hand, the employment of ELISA assays based on recombinant proteins reduces the inter-assay variability and the issues associated with antigen supply; nonetheless, the identification and production of S. stercoralis recombinant immunogenic proteins is costly and the development of a high-performance immunoassay is challenging and timeconsuming.
The utility of S. ratti somatic larval crude extract is well recognized, since it is employed in a commercial serological ELISA kit used for the diagnosis of human strongyloidiasis (Strongyloides ratti IgG ELISA, Bordier Affinity Products, CH). S. ratti ESPs have also been reported as immunoreactive with serum from infected rats as well as with serum from patients with human strongyloidiasis, and this immunoreactivity was shown to be life stage-dependent, since Pf-and iL3-ESPs displayed stronger reactivity compared to Fl-ESPs [17].Even though only a limited number of samples was investigated and only total IgG antibodies were measured, Soblik and colleagues provided the first evidence of ESP immunoreactivity with patients' serum, further supporting the importance of this antigenic fraction at the host-parasite interface.
As previously mentioned, S. venezuelensis has also been evaluated in a number of studies as a heterologous source of immunogenic antigens.The immunoreactivity of S. venezuelensis iL3 somatic protein extract (both soluble and membrane fractions) was evaluated using immunoblotting coupled with protein identification of immunoreactive bands.A 30-40 kDa immunoreactive antigenic fraction was shown to contain metabolic enzymes, cytoskeletal proteins and galectins [32].However, also based on previous results from S. ratti, the potential of ESPs as a source of immunoreactive antigens soon became clear and in 2017 Cunha and colleagues published the first report evaluating the utility of employing S. venezuelensis ESPs [57].Importantly, through a direct comparison they showed for the first time the superior immunoreactivity of S. venezuelensis ESPs compared to the somatic extract when tested with serum from infected patients and uninfected controls.ESPs showed lower crossreactivity with serum of patients suffering from other helminthiases compared to the somatic extract and was able to detect antibodies in both immunocompetent and immunocompromised Strongyloides-positive individuals [57].Those results were further confirmed by Roldán Gonzáles et al. [58], who compared the diagnostic accuracy of three different antigenic preparations from S. venezuelensis, namely soluble somatic fraction, membrane somatic fraction and ESPs.Once again, when tested for immunoreactivity with human serum, ESPs showed the highest accuracy for discriminating between Strongyloides-positive sera and negative controls, while displaying the lowest rate of cross-reactivity with serum from patients suffering from other helminthiases.Even though those comparative studies proved the higher potential of the ESP fraction for serodiagnosis, they also highlighted the need to identify by mass spectrometry the specific immunogenic antigens in relation to the development of novel diagnostic tools.Even if superior in accuracy compared to somatic extracts, the use of 'crude' ESPs in a diagnostic tool is still limited by the variability of the antigenic source, the need for animal models as a source of parasites and the lack of standardized procedures for ESP collection.Indeed, ESPs are obtained through the maintenance of larvae in vitro, and different maintenance conditions-in terms of incubation time, medium and larval density-have been reported in the literature.This technical variability seems to affect the larval metabolic state and, as a consequence, the ESP pattern [58] since differences in ESP proteomics composition were observed in larvae maintained in PBS or RPMI [27].The identification of immunoreactive proteins thus seems essential to overcome this variability, as was done in iL3-ESP from S. venezuelensis.Mass spectrometry in fact identified arginine kinase as a protein band that is highly immunoreactive with patients' serum (93% sensitivity and 100% specificity) and that has no cross-reactivity with serum samples from patients with other helminthiases [27].
An ideal serological test should however be based on homologous antigens in order to maximize both sensitivity and specificity.The first studies investigating the immunerecognition of proteins from S. stercoralis somatic extract by patients' serum date back to the '90s, when the detection of different polypeptide bands derived from S. stercoralis infective larvae by patients' antibodies was shown, even though band detection was not followed by protein identification [59][60][61].The first protein identification of S. stercoralis immuno-reactive bands using LC-MS/MS was performed by Rodpai and colleagues, who identified a 26 kDa band (corresponding to 14-3-3) and a 29 kDa band (corresponding to ADP/ATP translocase 4) from iL3 as highly immunogenic since they were recognized by patients' serum with SE/SP of 90%/76.5% and 80%/92.2%,respectively [29].However, only a limited number of clinical samples were tested (i.e.n = 10) and both bands presented cross-reaction, although limited, with other parasitic infections.The same research group later employed two-dimensional gel electrophoresis (2-DE) royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20220447 combined with immunoblot and protein identification by mass spectrometry to partly corroborate those findings.Indeed, using a more extensive separation technique they confirmed the immunogenic nature of 14-3-3 and detected additional protein spots as immunoreactive with patients' serum, among which galectin and enolase are worth mentioning [30].Both these proteins have already been identified in multiple studies as candidate diagnostic markers both in Strongyloides spp. as well as in other parasitic helminths [17,27,28].Moreover, enolase has already been proposed as a vaccine candidate against Ascaris suum [62], while galectins are involved in the initiation of the host immune response [35,[63][64][65].
In silico approaches nowadays represent important alternatives for the prediction of potentially immunogenic proteins, and immuno-informatics can be efficiently employed to screen larger protein datasets that are now available, either as predicted by the draft genomes or as experimentally obtained.Recently, such an approach has also been applied to S. stercoralis.In particular, a reverse approach using different algorithms and web-based tools was employed by Culma [31] to predict potentially immunogenic peptides from the UniProt S. stercoralis proteome (12.851 entries).The adopted pipeline, encompassing prediction of subcellular localization, helices, allergenicity, B-and T-cell epitopes, and evaluation of homology and physicochemical properties, highlighted 34 proteins as the most promising candidates for the future development of vaccines or diagnostics.Interestingly, none of these proteins was found by our group in a recent study using different B-cell epitope prediction tools to highlight potentially immunogenic proteins from S. stercoralis iL3 somatic proteome [21].This apparent discrepancy could be explained by the fact that in our study we focussed the prediction analysis only on proteins experimentally identified from iL3 larvae, while Culma used the entire proteome of S. stercoralis available in UniProt, encompassing proteins from all developmental stages [31].Importantly, among the candidates proposed in our study we highlighted galectin, confirming previous results [30] and Ss-NIE.This latter protein is currently employed in the most promising serological assay based on recombinant proteins [22][23][24][25].

Proteomics to study the host response to the infection
The evaluation of the host response through the high-throughput analysis of a host's body fluids or tissues during strongyloidiasis has yet to be performed.Such analysis could however be particularly useful in revealing novel facets of the pathological mechanisms associated with human strongyloidiasis, especially when applied in comparative studies.Indeed, this strategy has already been successfully applied to the study of other helminthiases, including schistosomiasis, fasciolosis and echinococcosis, to identify proteins or biological processes altered in the pathological state compared to uninfected controls, during the disease progression or in response to treatment [66][67][68][69][70][71][72][73][74][75][76].The most commonly investigated samples are plasma/serum and plasma/serumderived EVs (either from animal models or from patients), even though also tissue proteomics has been described [69,70].Importantly, a number of studies identified parasite proteins within the host's systemic or vesicular proteome, supporting the relevance of host proteomics also in revealing novel diagnostic markers [67,68,71,72,74,75]. Considering strongyloidiasis, many studies have investigated the systemic host response to the infection through the measurement of specific circulating factors, especially cytokines [11,[77][78][79][80][81][82][83].However, an untargeted high-throughput approach has yet to be undertaken.
In our group, we recently employed SWATH-MS proteomics to identify and quantify serum proteins from n = 5 patients suffering from long-lasting chronic strongyloidiasis (electronic supplementary material).The investigated population represented a sub-group of a cohort of elderly Italian subjects suffering from autochthonous strongyloidiasis that was previously studied to evaluate the systemic levels of selected cytokines, chemokines and immune factors [11].Here, we sought to expand those investigations through the untargeted analysis of the systemic proteome using LC-MS/MS.Serum samples taken on admission (baselinebefore treatment) and 6 and 12 months post-treatment with ivermectin were analysed, together with samples from ageand gender-matched uninfected controls (electronic supplementary material, tables S1 and S2).All patients whose serum was analysed signed a written informed consent for the donation of their biological samples for research purposes.The study received ethical approval by the Ethical Committee of Verona and Rovigo provinces under the protocol no.23730 (24 April 2019).
Our analysis quantified 208 serum proteins, 17 of which were found to be significantly differentially abundant between infected subjects at baseline compared to uninfected controls (i.e.p-value < 0.05; fold change, FC ≤ 0.83 or FC ≥ 1.2).Considering post-treatment samples, 9 and 8 proteins were significantly differentially abundant at 6 and 12 months after treatment compared to baseline, respectively (figure 1a,b; electronic supplementary material, table S3).Among differential proteins, coagulation factor 5 (FA5) was significantly increased in infected patients compared to controls, and its profile reverted with a significant decrease in abundance both 6 and 12 months post-treatment (figure 1c).Cyclin-dependent kinase 10 (CDK10) and Ubinuclein-1 (UBN1) showed increased serum abundance after treatment (both at 6 and 12 months) compared to baseline, while epidermal growth factor (EGF)-containing fibulin-like extracellular matrix protein 1 (FBLN3) decreased after treatment (figure 1c).It is noteable that 3 proteins associated with blood coagulation and platelet activation (namely FA5, von Willberand factor and fibrinogen) were significantly increased in infected patients compared to controls.Despite this observation, the overall number of modulated proteins was not large enough to allow performance of a formal pathway analysis to highlight potentially modulated biological processes associated with chronic longlasting strongyloidiasis.This could suggest a fine-tuning modulation in protein expression occurring at the systemic level in patients infected for such a long time, in agreement with the establishment of mechanisms of disease tolerance in these patients [12], allowing the infection to persist without developing a significant pathology.In order to highlight these small changes in protein abundances, a larger number of samples should probably be analysed and a comparison with samples from patients from endemic areas should be performed.Such patients should in fact bear infections of more recent acquisition and should probably be more frequently re-exposed to the pathogen.We did not identify any parasite royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20220447 proteins in our samples, but this is not surprising since at this chronic stage of infection parasites are confined to the intestinal tract, with females reproducing by parthenogenesis within the intestinal mucosa [9].
If extended to a larger number of samples (including other patient groups) and/or to other matrices, such as serum-derived EVs, host proteomics analyses might reveal novel aspects associated with the pathophysiology of chronic strongyloidiasis that will be fundamental to understanding the mechanisms of maintenance of parasitism and tolerance.

Conclusion and future perspectives
Proteomics studies of Strongyloides spp.have for long time been limited by the lack of reference or draft genomes, which have only been available since 2016 [19].The availability of high-quality data of helminth genomes is supporting and accelerating large-scale gene expression studies using 'omics approaches, including proteomics.Indeed, proteomics approaches can be particularly useful in studying different aspects of helminthiases of clinical importance, including: (i) to gain novel insights into parasite biology; (ii) to discern the mechanisms of host-parasite interaction; (iii) to identify novel diagnostic and therapeutic targets as well as potential vaccine candidates.An important limitation associated with S. stercoralis proteomics is however the lack of a manually annotated reference proteome.Indeed, the reference proteome currently available in UniProtKB/TrEMBL contains 12900 entries (organism ID 6248, as of 18th of July 2023) corresponding to unreviewed protein sequences automatically annotated.In order to make comparative proteomics data more trustworthy and impactful, it is still necessary to improve current annotation at both  Working with Strongyloides spp.presents other significant challenges mainly related to their complex life cycles.The investigation of clinical isolates, obtained either from patients' samples or from wild animals, should probably be favoured over laboratory strains maintained in laboratory animals.It has in fact been hypothesized that laboratory strains may not be entirely representative of the parasite's behaviour, as the different environments are likely to impact on their biological features and developmental strategies [84].In the post-genomics era, parasitology research has significantly grown, bringing our knowledge of parasite biology and the mechanisms of host-parasite interaction to a new level.Pioneering work has been done on Strongyloides spp., using CRISPR/Cas9-mediated mutagenesis, for instance [85], and this nematode still remains the most studied model for genetic transformation [86,87], highlighting the potential of this parasite as a model nematode to study the mechanisms of parasitism.Nonetheless, the potential differences between clinical/wild isolates and laboratory strains should be kept in mind and both comparative and functional studies should thus be focussed as much as possible on clinical/ wild isolates.
The complex life cycle of Strongyloides spp.still hampers the in vitro growth of this parasite.Although ambitious, the development of an in vitro model able to reproduce the parasitic life cycle would represent a major breakthrough in Strongyloides research, as it would provide the proper amount of parasitic material to perform in depth 'omics and functional studies while reducing the use of laboratory animals.
Large proteomics datasets are now being made available for different Strongyloides spp., for different life stages and for different fractions.Even though there are still some gaps to be filled, it is now necessary to bring proteomics studies a further step forward through functional analysis of specific targets of interest.Indeed, we should take advantage of Strongyloides as a nematode model for genetic transformation to explore the mechanisms of parasitism, of host-pathogen interaction and of host response at the molecular level in order to obtain novel tools to fight strongyloidiasis.
Ethics.All patients whose serum was analysed signed a written informed consent for the donation of their biological samples for research purposes.The study received ethical approval from the Ethical Committee of Verona and Rovigo provinces under the protocol no.23730 (24 April 2019).
The data are provided in electronic supplementary material [89].
Declaration of AI use.

Figure 1 .
Figure 1.Results of host serum proteomics analysis applied to investigate samples from patients with strongyloidiasis (taken before treatment (BT) and 6 and 12 months after treatment) and uninfected controls.For each group n = 5 samples were analysed.(a) Volcano plot showing the differential abundance of the quantified proteins in the different samples.The dotted line on the y-axes represents 0.05 p-value threshold, while the dotted lines on the x-axes represent 0.83 and 1.2 FC thresholds.(b) (overleaf ) Table summarizing proteins significantly differentially abundant in at least one of the three analysed comparisons.For each protein, the p-value and the fold change are reported.Orange values: p-value < 0.05; blue values: FC ≤ 0.83 or ≥1.2.(c) Detailed results for four proteins showing differential abundance in at least two of the assessed comparisons.The bar charts show the mean normalized area values for each group; error bars represent the standard error.Grey, uninfected controls; pink: patients with strongyloidiasis.All plots were generated using GraphPad Prism 8 (GraphPad Software LLC, MA, USA).

Table 1 .
Summary of published studies dealing with proteomics investigations of Strongyloides spp.Pf, parasitic female; Ff, free-living female; Fl, free-living larva; HSPs, heat shock proteins. a Table summarizing proteins significantly differentially abundant in at least one of the three analysed comparisons.For each protein, the p-value and the fold change are reported.Orange values: p-value < 0.05; blue values: FC ≤ 0.83 or ≥1.2.(c) Detailed results for four proteins showing differential abundance in at least two of the assessed comparisons.The bar charts show the mean normalized area values for each group; error bars represent the standard error.Grey, uninfected controls; pink: patients with strongyloidiasis.All plots were generated using GraphPad Prism 8 (GraphPad Software LLC, MA, USA).royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20220447 We have not used AI-assisted technologies in creating this article.All authors gave final approval for publication and agreed to be held accountable for the work performed therein.Conflict of interest declaration.We declare we have no competing interests.